Clinical and genetic characteristics of Chinese patients with congenital cranial dysinnervation disorders

Background Congenital cranial dysinnervation disorders (CCDDs) are a group of diseases with high clinical and genetic heterogeneity. Clinical examinations combined with Magnetic resonance imaging (MRI) and whole exome sequencing (WES) were performed to reveal the phenotypic and genotypic characteristics in a cohort of Chinese CCDDs patients. Results A total of 122 CCDDs patients from 96 families were enrolled. All patients showed restrictive eye movements, and 46 patients from 46 families (47.9%, 46/96) were accompanied by multiple congenital malformations. Multi-positional high-resolution MRI was performed in 94 patients from 88 families, of which, all patients had hypoplasia of the cranial nerves except HGPPS patients and 15 patients from 15 families (17.0%,15/88) were accompanied by other craniocerebral malformations. WES was performed in 122 CCDDs patients. Ten pathogenic variants were detected in KIF21A, TUBB3, and CHN1 genes in 43 families. Three variants were unreported, including KIF21A (c.1064T > C, p.F355S), TUBB3 (c.232T > A, p.S78T) and CHN1 (c.650A > G, p.H217R). Of the 43 probands harboring pathogenic variants, 42 were diagnosed with Congenital Fibrosis of Extraocular Muscles (CFEOM) and one was Duane Retraction Syndrome (DRS). No definite pathogenic variants in known candidate genes of CCDDs were found in sporadic DRS, Möbius Syndrome (MBS) and Horizontal Gaze Palsy with Progressive Scoliosis (HGPPS) patients. The CFEOM patients harboring R380C, E410K and R262H variants in TUBB3 gene and F355S variant in KIF21A gene exhibited syndromic phenotypes. Conclusions This study broadened the phenotypic and genotypic spectrums of CCDDs, and it was the largest clinical and genetic investigation for CCDDs patients from China. KIF21A and TUBB3 were the common pathogenic genes in Chinese CFEOM. MRI coupled with WES can provide a supportive diagnosis in patients with clinically suspected CCDDs. Supplementary Information The online version contains supplementary material available at 10.1186/s13023-022-02582-5.


Clinical and MRI evaluation results of CCDDs
A total of 122 Chinese self-reported Han patients from 96 not known to be related families with CCDDs were enrolled in the study (24 familial and 72 sporadic; age ranged from 5 months to 60 years); their demographic data and systemic features are provided in Table 1
The protein spatial structure of KIF21A was predicted by SWISS-MODEL, and F355 is located in the possible protein binding pocket (Fig. 4A, B). Force analysis showed a weakened interaction force between the amino acid residues at F355 and the surrounding amino acid residues after the mutation (Fig. 4C, D). The free energy analysis indicated that the free energy of the whole structure changed greatly, and the stability of the protein structure decreased after the mutation ( Table 2).
The CHN1 protein structure came from PDBID:3CXL, and H217 is located in the binding pocket of the spatial structure (Fig. 4F, G). Force analysis showed a weakened interaction force after the mutation (Fig. 4H, I). The free energy analysis indicated that the free energy changed greatly, and the stability of the protein structure decreased after the mutation, suggested that H217 is a key site to maintain the structural stability of CHN1 ( Table 2).
The TUBB3 protein structure came from PDBID:7PJF, and S78 is located in the surface of spatial structure (Fig. 4K, L). No obvious change in interaction force was found after the mutation (Fig. 4M, N). No obvious change in the free energy was found after the mutation, indicated that the structural stability remains unchanged after mutation (Table 2).
Among the 43 patients harboring variants, 42 were diagnosed with CFEOM and one was familial DRS. No potential pathogenic variants in known candidate genes of CCDDs variant were found in sporadic DRS and MBS. For HGPPS, more than ten heterozygous variants were found in ROBO3 gene, according to American College of Medical Genetics and Genomics (ACMG) standards, no variants were classified as pathogenic or likely pathogenic.

KIF21A
Four variants (R954W, R954Q, M947T and F355S) in KIF21A gene were identified in 31 CFEOM families. The two most common variants were R954W (77.4%, 24/31) and R954Q (16.1%, 5/31), and the other two less common variants were M947T (3.2%, 1/31) and F355S  was also accompanied by congenital distal joint contracture, and the patient C46 exhibited facial weakness, frontal bony prominence, delayed developmental milestones and thinning of corpus callosum in MRI (Fig. 5). Furthermore, we also observed that patients harboring the same KIF21A variants may also have different severity of CFEOM, even if they come from the same family.

TUBB3
Five variants (R262C, R262H, E410K, R380C and S78T) were identified in TUBB3 gene in 11 CFEOM families. The two most common variants were R262C (54.5%, 6/11) and E410K (18.2%, 2/11), less commonly, R380C (9.1%, 1/11), R262H (9.1%, 1/11) and a novel variant S78T (9.1%, 1/11). Clinical and genetic findings of the 11 CFEOM probands with TUBB3 variants were listed in Table 3. The patients harboring R262C and S78T showed milder phenotypes compared to the patients with R380C, E410K and R262H variants. The majority of R262C patients and one S78T patient showed isolated CFEOM, except patient C65 (R262C) who showed the absence of  Table 3). Facial weakness and hypogonadotropic hypogonadism occurred in the patients harboring E410K and R262H variants. Psychomotor retardation occurred in patients with R380C, E410K and R262H variants. The patient carrying the R380C variant had delayed developmental milestones, and he was given rehabilitation treatment for 3 months at the age of 2. At the age of 3, he could walk with the help of others. When walking, his legs were not straight, and he had impaired gait.
Common features of MRI from patients with R380C, R262H and E410K variants were malformations of the corpus callosum, anterior commissure and lateral ventricle. Malformations of basal ganglia, thalamus, brainstem and cerebellar were correlated with specific variants-R380C and R262H variants. Absence of facial nerves (CN7) and dysplasia of olfactory bulbs and sulci (OBS) were correlated with R262H and E410K variants (Figs. 6, 7).

DRS
A total number of 11 DRS patients from 8 families (2 familial and 6 sporadic) were enrolled, and they all exhibited congenital esotropia with limited horizontal eye movement accompanied by globe retraction which results in narrowing of the palpebral fissure. MRI revealed hypoplasia of bilateral abducens nerves (CN6) and dysinnervation of CN3.
No pathogenic variants were identified in sporadic DRS cases. Only one pedigree (D2) found a novel variant (c. 650A > G, p. H217R) in CHN1 gene. The patient D2-2 had congenital esotropia, nystagmus, limited horizontal eye movements and globe retraction with narrowing of the palpebral fissure on attempted adduction. The patient had classic DRS and additional vertical movement anomalies, with the right adducted eye slightly depressed and the left adducted eye slightly elevated. MRI revealed hypoplasia of CN6 and aberrant innervation of the lateral rectus (LR) by the inferior branches of CN3 (Fig. 8).

MBS and HGPPS
The study enrolled 17 MBS and 5 HGPPS patients. All MBS patients were sporadic and showed congenital, nonprogressive abduction deficits and facial weakness. MRI revealed hypoplasia of CN6 and CN7 in all MBS patients ( Fig. 9). Notably, nine MBS patients (52.9%, 9/17) had intrauterine exposure to adverse factors.
All HGPPS patients were from families of non-consanguineous marriage and showed absence of conjugate horizontal eye movements with intact vertical eye movements, accompanied by variable degrees of scoliosis (Fig. 10). Among four patients who underwent MRI, all had the typical radiographic features of HGPPS, including a deep posterior midsagittal cleft of the pons and butterfly-like morphology in the medulla.
No definite pathogenic variants in known candidate genes were identified in MBS and HGPPS patients.

Discussion
In this study, we combined MRI and WES to reveal and broaden the phenotypic and genotypic spectrums in 122 Chinese CCDDs patients from 96 families.
KIF21A and TUBB3 were the common pathogenic genes of Chinese CFEOM The mutation detection rate of CFEOM was the highest (63.6%, 42/66), which was much higher than other disease groups (DRS, MBS and HGPPS  [22]. These results are consistent with our current research findings. KIF21A belongs to a family of kinesin motor protein, which involved in cargo transport along microtubules [5]. Pathogenic variants in KIF21A gene may cause CFEOM by modifying the autoinhibitory interaction between the motor domain and a regulatory region in the stalk [23]. The majority of previously reported variants located in coiled coil domains of KIF21A, meanwhile, the CFEOM patients are almost all present with isolated phenotype [5]. However, the novel variant F355S identified in our study was located in conserved N-terminal motor domain, and the patient showed rare syndromic phenotype. To our knowledge, only three variants located in the motor region of KIF21A have been reported, they were C28W, D352E, M356T [5,24,25]. The patients harboring C28W and M356T variants showed isolated CFEOM, while the patients harboring D352E variant present with syndromic phenotype. Notably, patient harboring F355S in this study and the patient harboring D352E had the similar phenotypes, including CFEOM, facial weakness, frontal bony prominence, exposure keratitis and delayed developmental milestones. The difference is that the F355S patient also had the hypoplasia of corpus callosum in MRI. We speculate that the F355S may attenuate autoinhibition to a greater degree, or induce disease via different pathways [26].
TUBB3 gene encodes the neuron-specific beta-tubulin isotype 3 which plays a critical role in axon guidance and neuronal migration. Pathogenic variants in TUBB3 may cause CFEOM by altering dynamic instability of microtubules or disrupting the interaction of microtubules with kinesin motors [27].
We found a novel variant TUBB3-S78T in a CFEOM pedigree, the proband has normal developmental history (C9-2). The S78T is located in the N-terminal domain of TUBB3, so far, several variants have been reported in the region of S78. Whitman et al. [28] reported the patients harboring G71R and G98S variations presented with syndromic phenotypes, manifested as CFEOM, nystagmus, torticollis, developmental delay, intellectual and social disabilities. MRI showed the hypoplasia of the corpus callosum and anterior commissure, malformations of hippocampi, thalami, basal ganglia and cerebella, and hypoplasia of brainstem and cranial nerve. The patients harboring the S78T variant in this study and the patient harboring R62Q variant reported by Max A. Tischfield in 2010 presented with isolated CFEOM [6]. Although the S78T patient (C9-2) underwent MRI exam of the ocular motor nerves, and had the normal corpus callosum configuration, however the high resolution brain MRI data was absent, so we cannot rule out the possibility that he has subtle cortical dysplasia. In addition, we noted that G82R have been reported to cause MCD without CFEOM [29].
Compared to patients harboring KIF21A variants, the CFEOM patients with TUBB3 variants tend to show syndromic phenotypes. In the past, researchers named the disease caused by the TUBB3 variants as the "TUBB3 Syndromes" [6]. In recent years, researchers found that different variants of TUBB3 usually produce different syndromic phenotypes [6]. In our cohort, the patients harboring E410K and R262H variants of TUBB3 gene exhibited CFEOM and accompanied by different multiple congenital malformations, and their clinical and MRI findings were highly similar to previous reports [4,30], which confirmed that the correlations between specific variants and phenotypes were strong. Therefore, researchers preferred to name these syndromic phenotypes after specific variants. Up to date, "TUBB3 E410K syndrome", "TUBB3 R262H syndrome" and "TUBB3 M323V syndrome" were used to name the diseases caused by E410K, R262H and M323V variants, respectively [4,31,32]. Furthermore, the patients with "TUBB3 E410K syndrome" and "TUBB3 R262H syndrome" were easily misdiagnosed as MBS owing to similar clinical manifestations (facial weakness and limitation of eye movements etc.) [33]. MRI, WES and specific general examinations based on the good phenotype-genotype correlations can contribute to accurate diagnosis and follow-up.
TUBB3 R380C variant was identified in one CFEOM patient (C6) in our cohort, and this variant has been previously reported only once in a CFEOM patient [6]. The clinical and MRI findings of these two patients were highly similar, both of them manifested CFEOM with intellectual impairment, multiple congenital craniocerebral malformations which involved in commissural fiber, brainstem, basal ganglia, thalamus, cerebral ventricle and cerebellum etc. However, "bilateral chest wall malformation" and impaired gait were observed only in our R380C patient. Previous research indicated sensorimotor polyneuropathy occurred in individuals harboring R262H, E410K and D417H/N [6]. We speculated that the R380C patient in this study may have similar sensorimotor polyneuropathy, and electromyography results are needed to confirm the suspected diagnosis.
Furthermore, we also noted that TUBB3 R380C variant had been reported in patients diagnosed with "neurodevelopmental disabilities" and "cerebellar dysplasia (CD)" [34,35], however the ocular manifestations of the patients were not described clearly by authors.
In present study, three patients with TUBB3 E410K and R262H variants (C21, C32 and C52) fitted the diagnostic criteria of Kallmann syndrome, confirmed the previous report results and suggested that TUBB3 gene may involve in the pathogenesis of Kallmann syndrome [4].

Genetic screening results of sporadic DRS, MBS and HGPPS cases were poor
DRS is a congenital motility disorder which characterized by restricted abduction or adduction and accompanied by globe retraction and narrowing of the palpebral fissure [36]. CHN1 gene was a common causative gene for familial non-syndromic DRS, which was identified in approximately 35% of familial DRS cases [11]. Functional analysis revealed that CHN1 variants caused CN6 growth stalled, leading to secondary dysinnervation of the LR by the CN3 [37]. To date, variant detection rate was poor in sporadic isolated DRS patients, so genetic screening is suggestive for those individuals with positive family history Fig. 6 Clinical and MRI spectrum of CFEOM patients with TUBB3 variants. (a1-a3), Patient C9-2 harboring S78T variant. a1 Congenital ptosis of right eye. a2 Atrophy of levator palpebrae superioris-superior rectus (arrow) in right eye. a3 Hypoplasia of the CN3 at the brainstem. The MRI was taken at the age of 4y. b1-b3 Patient C65 harboring R262C variant. b1 Congenital bilateral ptosis. b2, b3 Hypoplasia of CC and CN3. The MRI was taken at the age of 1y. c1-c6 Patient C6 harboring R380C variant. c1, c2 Congenital bilateral ptosis and chest wall malformation (red box). c3, c4 Hypoplasia of CC, brainstem (c, star), cerebellum (CER), basal ganglia (BG), thalamus (THA) and CN3 with the enlargement of lateral ventricle (LV). The MRI was taken at the age of 11y. d1-d8 Patients harboring E410K variants. d1, d2 Facial pictures of two patients (C21 and C32). d3-d8 Patient C32 exhibits hypoplasia of CC (d3), asymmetry of LV (d4), absence of olfactory bulbs and sulci (d5, arrow), hypoplasia of CN3 (d6) and absence of bilateral CN7 (d7, d8, arrow). The MRI was taken at the age of 19y [38]. In our DRS cohort (2 familial and 6 sporadic), we identified one novel variant H217R in CHN1 gene in a large DRS family, no causative genes were identified in all sporadic DRS cases and one DRS family. These results were similar to the previous study [38].
MBS is a rare disease with minimum diagnostic criteria of congenital limitation of ocular abduction and facial weakness [3]. In 2015, Laura Tomas-Roca et al. reported that PLXND1 and REV3L may be pathogenic genes for MBS, and animal researches indicated PLXND1 and  (d, star), CER, BG, THA with asymmetry of Sylvian fissure (SF) and LV. g-j Absence of olfactory bulbs and sulci (g, arrow), CN3 (h, arrow) and CN7 (i, j, arrow). The MRI was taken at the age of 4y  REV3L may cause a defect in the facial branchiomotor neuron migration [17]. This was the first report of pathogenic gene for MBS, however, these two genes have not been reported again in other MBS cases to date. In addition to genetic factors, intrauterine environmental factors were also reported be involved in MBS [39]. In our study, no causative gene was identified in all MBS patients, and more than half of the patients had intrauterine exposure to adverse factors. These results suggested that multifactorial pathogenic mechanism maybe exist in MBS.
HGPPS is a rare autosomal recessive disorder characterized by severe restriction of conjugate horizontal eye movements and progressive scoliosis [16]. Reviewed the published reports about HGPPS, it could be found that ROBO3 was the pathogenic gene and the HGPPS patients harboring ROBO3 variants often existed parental consanguinity [40,41]. ROBO3 plays an important role in axonal guidance which mediate midline crossing of neurons during embryogenesis of the spinal cord [16]. Our study enrolled five sporadic HGPPS patients who were from non-consanguineous family. No homozygous variants of ROBO3 gene were found, and the possibility of compound heterozygous mutation could not be ruled out. More than a dozen heterozygous variants of ROBO3 gene were identified in five HGPPS patients, however, no variants were classified as pathogenic or likely pathogenic according to ACMG standards, so the pathogenicity of these variants cannot be determined.
The mutation detection rates in sporadic isolated DRS, MBS and non-consanguineous families with HGPPS were poor in this study. The reasons could be multiple, such as epigenetic factors, environmental or other nongenetic factors may be involved in the pathogenesis of diseases.
The phenotypes of CCDDs are diverse and complex, often involving multiple tissues and organs, many new MRI and clinical phenotypes have been reported and discovered. Face to face communication with radiologist is necessary. At the same time, the participation of clinical multidisciplinary doctors is required to carry out professional examinations, diagnosis and treatment. Considering these facts, future efforts need to focus on mining, refining and quantifying extraocular manifestations to improve our understanding of clinical and genetic basis of CCDDs. Meanwhile, the novel variants need future functional studies to determine its pathogenicity.

Conclusions
This study was the largest clinical and genetic research for CCDDs patients from China, the results broadened the phenotypic and genotypic spectrums of CCDDs. KIF21A and TUBB3 were the common pathogenic genes of Chinese CFEOM. Compared with patients with KIF21A variants, patients with TUBB3 variants have better genotype-phenotype associations. The patients harboring R380C, E410K and R262H variants in TUBB3 gene and F355S amino acid variant in KIF21A gene exhibited syndromic phenotypes. Genetic screening results of sporadic DRS, MBS and HGPPS cases were poor. These findings were highly consistent with previous results. The combination of MRI and WES was effective for accurate and supportive diagnosis of clinical suspected CCDDs, and the research results would be valuable for future gene therapy and facilitate further understanding of pathogenic mechanism of CCDDs.

Patients
This was a prospective cohort study with retrospective data analysis. A total of 122 self-reported Han patients from 96 not known to be related families with CCDDs were enrolled in the outpatient department of Beijing Tongren Hospital affiliated to Capital Medical University from January 2013 to December 2021.
The inclusion criterion was (1) congenital, non-progressive restrictive eye movements and/or dysplasia of the cranial nerves in MRI, or (2) congenital, non-progressive limitation of horizontal eye movements and progressive scoliosis, with or without dysplasia of the cranial nerves [1].
The study complied with the tenets of the Declaration of Helsinki and was approved by the Institution Review Board of Beijing Tongren Hospital, Beijing, China (TRECKY2018-035). All participants we enrolled gave written informed consent.

Clinical evaluation and MRI
All participants underwent a detailed ophthalmic and physical examinations. The ophthalmic examinations included cycloplegic refraction, best-corrected visual acuity (BCVA), binocular alignment, eye movements, measurement of the height of palpebral fissures and the function of levator palpebrae muscles, slit lamp examination and fundus examination.
Multi-positional high-resolution MRI was performed in 96 patients from 88 families with a General Discovery MR750 3.0-T Twinspeed scanner. Children under 10 years old were given oral chloral hydrate (10-15 mg/ kg) 30 min before examination. Imaging of cranial nerves at the brainstem was conducted with an 8HR brain coil and adopted the three-dimensional fast imaging employing steady-state acquisition (3D-FIESTA) sequence. Parameters were as follows: axial scanning; repetition time (TR), 4.28 ms; echo time (TE), 2.04 ms; flip angle (FA), 60°; the field of view (FOV), 16 × 16 cm; Matrix, 256 × 320; section thickness, 0.8 mm; number of excitation (NEX), 4. Orbital imaging was performed with an 8HR brain coil in oblique coronal planes and applied the following parameters: TR, 325 ms; TE, 9.38 ms; FOV, 17 × 17 cm; matrix, 264 × 256; section thickness, 3.0 mm; layer spacing, 0.3 mm; NEX, 2. Scanning of the brain structure was conducted with a phased array coil in head-cross section and the parameters were as follows: Fast spin-echo/T2-weighted images (FSE/T2WI); section thickness, 6.0 mm; layer spacing, 1.0 mm.

Whole exome sequencing and bioinformatics analysis
Whole blood samples were collected from all participants and their nuclear family members, genomic DNA was extracted using TianGen Biochemical DNA isolation kit (Beijing Tiangen Biochemical Co., Ltd, Beijing, China).
Whole exome sequencing was performed at Beijing Novogene Bioinformatics Technology Co., Ltd (Beijing, China). Agilent SureSelect Human All ExonV6 kit was used to capture whole exome sequence. Sequencing was performed on illumina HiSeq4000 platform using PE150 sequencing strategy.
Valid sequencing data was mapped to the reference genome (GRCh37/hg19) by BurrowsWheeler Aligner (BWA) software. Samtools and Sambamba were used to sort bam files. Samtools and bcftools were used to do variants calling and identified SNPs and indels. ANNOVAR was used to do annotation for VCF (Variant Call Format) file with a variety of databases, such as dbSNP, 1000 Genome, GnomAD, CADD and HGMD. Gene transcript annotation databases, such as Consensus CDS, RefSeq, Ensemble and UCSC, were also applied for annotation to determine amino acid alternation. Variants were filtered if MAF (minor allele frequency) > 1% in more than one of the four frequency databases (1000 genomic data, esp6500, gnomAD_ALL and gnomAD_EAS). Nonsynonymous variants were screened according to scores of SIFT, Polyphen, VariantTaster and CADD. CNVs analyses were also performed. ACMG guidelines were used to evaluate variants. After the candidate pathogenic variants (KIF21A, TUBB3, TUBA1A, TUBB2B, TUBB6,  PHOX2A, CHN1, MAFB, SALL4, HOXA1, HOXB1,  ROBO3, PLXND1, REV3L, COL25A1 and ECEL1) were found, Sanger sequencing was used for validation. Trios/pedigree analysis were performed to establish cosegregation or de novo status.